Cervical carcinomas are associated with a few specific subtypes of the human papillomavirus (HPV). However, the molecular mechanisms of cellular transformation by these "high-risk" HPV are not fully understood. Two transforming oncoproteins of HPV, E6 and E7, are required for both initiation and maintenance of cellular transformation. The transforming function of E6 is mediated partially by its ability to induce ubiquitination and degradation of the p53 tumor suppressor protein. However, E6 also has poorly understood transformation activities that are p53-independent. These additional E6 activities have critical roles in the pathogenesis of cancers caused by HPV. Recently, we cloned and characterized a novel human E6-binding protein, MAML1 (mastermind-like-I) that selectively interacts with E6 from high-risk HPV type 16. MAML1 is related to the Drosophila melanogaster mastermind gene, a component of the Notch signaling pathway involved in cell fate determination. We found that MAML1, a nuclear protein, functions as a transcriptional co-activator for mammalian Notch receptors. These studies have now led to our cloning and characterization of two additional members of this novel mammalian mastermind family, MAML2 and MAML3. Like MAML1, both MAML2 and MAML3 preferentially bind to E6 from high-risk HPV16. Furthermore, high-risk HPV16 E6 proteins block Notch signaling. Therefore, the MAML family provides the first direct link between papillomavirus oncoproteins and the Notch pathway. The, goal of this proposal is to determine the mechanisms and significance of the HPV E6 interactions with the three mammalian mastermind family members in E6-mediated cellular transformation. The hypothesis to be tested is that HPV E6 interactions with the Notch pathway, via the MAML family, have a critical role in cellular transformation. Specifically, we hypothesize that E6-MAML interactions result in altered Notch signaling and lead to deregulated growth and differentiation in cervical epithelial cells. Alternatively or in combination, E6-MAML interactions may be required for E6 transformation activities involving cell proliferation, cell cycle regulation and senescence. To test these hypotheses, we will 1) determine whether the interactions of high-risk E6 proteins with MAML proteins are critical for E6 mediated cellular transformation, 2) determine whether and how the interactions of E6 and MAML proteins contribute to cellular transformation via Notch signaling. The proposed research will advance our understanding of the molecular mechanisms of transformation of cervical cancers, as well as provide insight into the novel MAML family and the Notch signaling pathway. [unreadable] [unreadable]